The aims of the proposed research are to gain a detailed thermodynamically rigorous understanding of the mechanisms by which protein structure, self-assembly and solubility are modulated by non- specific ligands, and of the linkages which exist between the binding of specific ligands and self-assembly reactions that impart to proteins their biological function and control it. Specifically, the following questions will be addressed. Establishing the mechanisms on the molecular level by which salting-out co-solvents are excluded from proteins, and the balance which exists between exclusion and binding that imparts to some salting-out agents a protein stabilizing ability, but not to others. Establishing quantitatively that stabilization is due to the balance between the preferential interactions of the co-solvent with the denatured and native states of the protein; to this end preferential interactions of the solvent components will be determined with thermally unfolded proteins. Establishing the role of the free energy of cavity formation in the thermodynamics of protein unfolding and its compensation by weak binding of co-solvents. Establishing the rules of additivity and compensation between binding and exclusion of co-solvents in mixed systems, such as a denaturant with a stabilizer, as has been found in nature in the case of osmolytes used by various organisms, and accounting thermodynamically for the selection by such organisms of a limited number of compounds to act as osmolytes or cryoprotectants, and in particular of "superosmolytes". Establishing of the controls by the nature of the nucleotide (GTP, GDP and analogues) that occupies the E-site on tubulin of the mode of self-assembly that tubulin undergoes (linear, such as sheets or microtubules, or curved, such as double rings). The methods used will be those of macromolecular physical biochemistry, such as sedimentation velocity and equilibrium, densimetry, differential refractometry, spectrofluorimetry, light scattering and melting curve spectrophotometry. A detailed understanding of the mechanisms by which solvent additives affect protein stability and solubility are important for the rational development of formulation by biotechnology and of cryoprotectants for organ preservation, as well as for the understanding of the selection by organisms of a small number of compounds to maintain osmotic pressure and protect them from damage by freezing. A characterization of the allosteric controls of tubulin self-assembly into structures of various geometries is important for the understanding of the dynamics of the tubulin-microtubule cycle in cellular functions such as transport and mitosis.